ILT3 Polypeptides and Uses Thereof

ABSTRACT

This invention provides a method for inhibiting the rejection of transplanted islet cells, comprising administering to the subject a polypeptide comprising all or a portion of the extracellular domain of ILT3, wherein the polypeptide is water soluble. This invention further provides a method of treating diabetes, by inhibiting the rejection of transplanted islet cells through the administration of the polypeptide to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority as a divisionalapplication under 35 U.S.C. §121 to U.S. application Ser. No.13/621,961, filed Sept. 18, 2012, which claims benefit of priority as adivisional application under 35 U.S.C. §121 to application Ser. No.12/419,824, filed Apr. 7, 2009, which is a continuation-in-part ofapplication Ser. No. 11/661,877, filed Mar. 2, 2007, which is a U.S.National Stage Application of PCT Appln. PCT/US05/31380, filed Sep. 1,2005, which in turn claims benefit from Provisional Applns. Nos.60/622,165, filed Oct. 26, 2004, and 60/607,095, filed Sep. 3, 2004. Thedisclosures of all applications are hereby incorporated in theirentirety herein.

STATEMENT OF GOVERNMENTAL INTEREST

The invention was made with government support under grants AI025210 andAI055234 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

Throughout this application, various documents are referenced. Fullcitations for these documents are presented immediately before theclaims. Disclosures of these documents in their entireties are herebyincorporated by reference into this application.

BACKGROUND LIRs

Leukocyte Ig-like receptors (“LIRs”) are a family of immunoreceptorsexpressed predominantly on monocytes and B cells and at lower levels ondendritic cells and natural killer (“NK”) cells. Activation of variousimmune cell types can be prevented by negative signaling receptorsthrough interactions with specific ligands, such as MHC 5 class Imolecules by NK cells. All of the LIR inhibitory receptors, members ofsubfamily B, contain a cytoplasmic immunoreceptor tyrosine-basedinhibitory motif (“ITIM”). Upon MHC class I (or other ligand) engagementand tyrosine phosphorylation of the ITIM, intracellular protein-tyrosinephosphatases such as SHP1 are recruited, and an inhibitory signalcascade ensues. Other LIR receptors, members of subfamily A, with shortcytoplasmic regions containing no ITIMs and with transmembrane regionscontaining a charged arginine residue, may initiate stimulatorycascades. One member of subfamily A lacks a transmembrane region and ispresumed to be a soluble receptor (1). LIR-5, one type of LIR, is alsoknown as immunoglobulin-like transcript 3 (ILT3).

ILT3 Fusion Proteins

A soluble fusion protein made of a soluble portion of ILT3 and the Fcportion of IgG1 is known. However, this fusion protein was used merelyas a negative control in an endotoxemia study, and its potential use asa therapeutic was not disclosed (2).

Soluble ILT4

LIR-2, also known as ILT4, is an inhibitory receptor However, itssoluble form was shown to completely restore the proliferation ofT-cells activated with LPS and IL-10-treated dendritic cells (3).

SUMMARY

This invention provides a method for treating islet cell transplantrejection in a subject who has received said transplant, comprisingadministering to the subject a therapeutically effective amount of afirst polypeptide comprising all or a portion of the extracellulardomain of ILT3, wherein the polypeptide is water-soluble and does notcomprise a Fc portion of an immunoglobulin, or a second polypeptidecomprising (i) all or a portion of the extracellular domain of ILT3operably affixed to (ii) the Fc portion of an immunoglobulin, whereinthe Fc portion of the immunoglobulin comprises a function-enhancingmutation, and wherein the polypeptide is water-soluble.

This invention further provides a method for treating a subjectafflicted with autoimmune diabetes, comprising administering to thesubject a therapeutically effective amount of the first or secondpolypeptide described above.

This invention further provides a method for treating a subjectafflicted with graft versus host disease, comprising administering tothe subject a therapeutically effective amount of the first or secondpolypeptide described above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic drawing of soluble ILT3 expression vector.

FIG. 2: Western Blot analysis of soluble ILT3 fusion protein.

FIG. 3A-3B: (FIG. 3A) Soluble ILT3 inhibits T lymphocyte proliferationin vitro. (FIG. 3B) Inhibition is abrogated by anti-ILT3 antibody.

FIG. 4A-4E: (FIG. 4A) Schematic diagram of MIG retroviral expressionvectors of ILT3 and ILT3 delta. (FIG. 4B-FIG. 4C) Fluorescencehistograms of ILT3 expression on the surface of KG1.ILT3 andKG1.ILT3delta. (FIG. 4D) Confirmation of the molecular weight ofILT3delta by Western Blot and determination of inability of ILT3deltamolecule to recruit SHP-1 by immunoprecipitation and Western blot. (FIG.4E) Inhibition of protein tyrosine phosphorylation in KG1.ILT3 but notin KG1.ILT3delta cells by crosslinking anti-HLA-DR and anti-ILT3 mAbs.

FIG. 5A-5E: (FIG. 5A) ILT3 and ILT3delta molecules inhibit proliferationof CD3+CD25− T cells in 5 day primary mixed leukocyte cultures (MLCs).(FIG. 5B) ILT3 and ILT3delta molecules suppress proliferation of CD4+ Tcells primed with KG1 cells in 3 day secondary MLC, which can bereversed by addition of IL-2 or anti-ILT3 mAb. (FIG. 5C-FIG. 5E) CD8+ Tcells primed with KG1, but not with KG1.ILT3 are cytotoxic to KG1 cellsat ET ratio of 1 to 1.

FIG. 6: siLT3 suppress proliferation of CD3+CD25− T cells in primary andsecondary MLCs.

FIG. 7: siLT3 inhibit generation CD8+ cytotoxic T cells.

FIG. 8: Frequency of IFN-y producing CD8+ T cells from T cells primedwith allogeneic APC with siLT3 is higher than CD8+ T cells primed withthe same APC without siLT3.

FIG. 9A-9E: (FIG. 9A) CD8+ T cells primed with allogeneic APC in thepresence of siLT3, but not in the absence of siLT3 suppressproliferation response of naive CD3+CD25− T cells from the sameresponder to the original stimulator. (FIG. 9B) CD8+ T cells primed withKG1.ILT3, but not KG1 suppress proliferation response of naive CD3+CD25−T cells from the same responder KG1 cells. (FIG. 9C) CD8+ T cells primedwith allogeneic APC with siLT3, but not without siLT3, or primed withKG1.ILT3 cells, but not with KG1 cells express high FOX3 protein inWestern blot analysis. (FIG. 9D-9E) CD8+ T cells primed with allogeneicAPC with siLT3, but not without siLT3 up-regulate ILT3 expression anddown-regulate CD86 expression on immature DC derived from the samestimulator for priming.

FIG. 10A-B: FITC-labeled siLT3 proteins stains allogeneic APC activatedCD4+ T cells at day 3 of primary MLC culture, but not activated CD8+ Tcells and naive CD4+ and CD8+ T cells.

FIG. 11: ILT3-Fc treatment prevents islet allograft rejection inhu-NOD/SCID mice. All of the mice in the treatment group (treated withITL-3-Fc; open circles) remained euglycemic (100% freedom from diabetes)through the study period of 91 days, indicating islet cell graftsurvival. By contrast, mice in the control group (IgG-treatment or notreatment; dark squares and dark circles, respectively) graduallyreturned to being diabetic (defined as having a blood glucose level>350mg/dl) over the course of 3-7 weeks (days 20-50), indicating a failureof islet cell graft survival.

FIG. 12: ILT3-Fc treatment prolongs the survival time of hu-NOD/SCIDislet allograft recipients independent of glycemia. Host survival timein ILT3-Fc and human IgG treated mice was calculated after exclusion ofmice that rejected the islet allograft and became diabetic. Dark lineindicates the percentage of surviving euglycemic mice receiving ILT3-Fctreatment (treatment group) over a period of time (0-91 dayspost-treatment). Light line indicates the percentage of percentage ofsurviving euglycemic mice receiving IgG treatment (control group).

FIG. 13A-13F: CD4+ or CD8+ T-cells sorted from mice killed on 23, 47 and91 days following humanization were added at increasing numbers(1-8×10⁴/well) to a fixed number (10⁴/well) of unprimed autologousCD3+CD25− T-cells and stimulated for 6 days in MLC with irradiated,allogeneic peripheral blood mononuclear cells (PBMC) sharing HLA-A, -B,and -DR antigens with the islet transplant. Cultures were harvestedafter 6 days and └3H┘ thymidine incorporation was measured to assayT-cell reactivity.

FIG. 14A-14B: Flow cytometry analysis of human PBMC engraftment in (FIG.14A)I LT3-Fc and human IgG treated (FIG. 14B) hu-NOD/SCID micerecipients of allogeneic islet cells. Mice were sacrificed on day 47.Splenocytes were stained with monoclonal antibodies raised against humanCD4, CD8, CD 14, CD19 and HLA.

FIG. 15A-15C: (FIG. 15A-FIG. 15B) Quantitative real-time PCR studies ofcytokines (IFN-y, IL-2, IL-5 and IL-10) expressed by human CD4+ and CD8+T cells isolated from ILT3-Fc and IgG treated, hu-NOD/SCID recipients ofislet allografts. Paired mice were sacrificed at week 4 and week 7. CD4+and CD8+ human T cells were sorted from the spleen and analyzed byreal-time PCR. (FIG. 15-C) Flow cytometry analysis of the expression ofT-cell activation markers CD28 and CD40L of human CD8+ T-cellscolonizing the spleen. Paired mice were sacrificed at week 4 and week 7and CD4+ and CD8+ human T cells were sorted from the spleen. Theexpression level of CD28 or CD40L was plotted against the expressionlevel of CD8, and the abundance of CD28+ and CD28−, as well as CD40L+and CD40L− cells was determined.

FIG. 16: Quantitative real-time PCR analysis of CD40 expression in humanpancreatic islet cells. Human pancreatic islet cells were cultured 1)alone, 2) with a mixture of inflammatory cytokines, 3) withCD40L-transfected D1.1 cells, 4) with D1.1 cells plus allospecific CD8+Ts cells, or 5) with D1.1 cells plus unprimed CD8+ T cells (n=3 for allculture conditions).

FIG. 17A-17F: Immunostaining of CD8 (FIG. 17A-FIG. 17D) and CD40 (FIG.17E and FIG. 17F) in sections of islet-engrafted kidneys fromILT3-Fc-treated (FIG. 17A, FIG. 17C, and FIG. 17E) and IgG-treated (FIG.17B, FIG. 17D, and FIG. 17F) NOD/SCID mice 23 days after humanization.

FIG. 18A-18D: Hematoxylin and eosin (H&E) staining in sections of lungtissue from ILT3-Fc-treated (FIG. 18A and FIG. 18C) and IgG-treated(FIG. 18B and FIG. 18D) islet allograft recipients, 23 days afterhumanization.

FIG. 19A-19D: H&E staining (FIG. 19A and FIG. 19B) and CD8immunostaining (FIG. 19C and FIG. 19D) in sections of islet-engraftedkidneys from ILT3-Fc-treated (FIG. 19A and FIG. 19C) and humanIgG-treated (FIG. 19B and FIG. 19D) hu-NOD/SCID mouse 47 days afterhumanization.

FIG. 20A-20F: Immunostaining of insulin in sections of islet-engraftedkidneys from ILT3-Fc-treated (FIG. 20A and FIG. 20C) and IgG-treated(FIG. 20B and FIG. 20D) hu-NOD/SCID mice 47 days after humanization.Insulin immunostaining (FIG. 20E) and hematoxylin-eosin staining (FIG.20F) in sections of islet-engrafted kidneys from ILT3-Fc-treatedhu-NOD/SCID mice 91 days after humanization.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present invention are generally performed according toconventional methods well known in the art. Generally, nomenclaturesused in connection with, and techniques of, cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are those wellknown and commonly used in the art. The methods and techniques of thepresent invention are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification unless otherwise indicated. See, e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates(1992, and Supplements to 2002); Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990); Principles of Neural Science, 4th ed., Eric R.Kandel, James H. Schwart, Thomas M. Jessell editors.McGraw-Hill/Appleton & Lange: New York, N.Y. (2000). The nomenclaturesused in connection with, and the laboratory procedures and techniquesof, molecular and cellular neurobiology and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or group of integers but not the exclusion of any otherinteger or group of integers.

Definitions

“Administering” shall mean delivering in a manner which is affected orperformed using any of the various methods and delivery systems known tothose skilled in the art. Administering can be performed, for example,topically, intravenously, pericardially, orally, via implant,transmucosally, transdermally, intradermally, intramuscularly,subcutaneously, intraperitoneally, intrathecally, intralymphatically,intralesionally, or epidurally. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

“Agent” shall include, without limitation, an organic or inorganiccompound, a nucleic acid, a polypeptide, a lipid, a carbohydrate or aphysical stimulus. Agents include, for example, agents which are knownwith respect to structure and/or function, and those which are not knownwith respect to structure or function.

“Concurrent” administration of two agents shall mean administrationwherein the time period over which the first agent is administeredeither overlaps with, or is coincident with, the time period over whichthe second agent is administered. For example, a first and a secondagent are concurrently administered if the first agent is administeredonce per week for four weeks, and the second agent is administered twiceper week for the first three of those four weeks. Likewise, for example,a first and second agent are concurrently administered if the first andsecond agent are each administered, in the same or separate pills, onthe same day, once per week for four weeks.

“Delaying” the onset of a disorder shall mean slowing the progression ofthe disorder, or extending the time before the onset begins.

“Engraftment” shall mean the incorporation of grafted (i.e.,transplanted) tissue or cells into the body of the host, or the processof transplanted stem cells reproducing new cells.

“Expression vector” shall mean a nucleic acid encoding a nucleic acid ofinterest and/or a protein of interest, which nucleic acid, when placedin a cell, permits the expression of the nucleic acid or protein ofinterest. Expression vectors are well known in the art.

“Extracellular domain of ILT3” shall mean the N-terminal 258 amino acidresidues of ILT3 (e.g., human ILT3 having the sequence of GenBankAccession No. U82979). A “portion” of the extracellular domain of ILT3includes, for example, the IgG1-like domain 1 (residues 42-102 of humanILT3), the IgG1-like domain 2 (residues 137-197 of human ILT3), and theN-terminal 250, 240, 230, 220, 210, 200, 190, 180, 170, 160 or 150 aminoacid residues of ILT3.

“Function-enhancing mutation”, with respect to the second polypeptide ofthis invention, shall mean any mutation which confers a physicalproperty (e.g., reduced binding of the Fc moiety to an Fc receptor) tothe polypeptide which permits it to better accomplish its therapeuticrole (e.g., through increasing its half-life or reducing adverse effectsotherwise caused by a subject's immune system).

“Humanization”, with respect to mice, shall mean the injection andsubsequent incorporation, or engraftment, of human haematopoietic stemcells or peripheral blood mononuclear cells (PBMC) into immunodeficientmice.

“Humanized mice” shall mean immunodeficient mice injected (i.e.,engrafted) with human haematopoietic stem cells or PBMC.

“ILT3” shall mean the gene, mRNA, or protein of “Immunoglobulin-LikeTranscript-3”, and is synonymous with “ILT-3”, “LIR-5”, “CD85K” and“LILRB4”. The mRNA coding sequence for human ILT3 is provided underGenBank No. U82979.

“Immunoglobulin” and “antibody” are used synonymously herein, and shallinclude, by way of example, both naturally occurring and non-naturallyoccurring antibodies. Specifically, this term includes polyclonal andmonoclonal antibodies, and antigen-binding fragments (e.g., Fabfragments, as opposed to Fc fragments) thereof. Furthermore, this termincludes chimeric antibodies (e.g., humanized antibodies) and whollysynthetic antibodies, and antigen-binding fragments thereof. Within thescope of the term “antibody” are also antibodies that have been modifiedin sequence, but remain capable of specific binding to an antigen.Example of modified antibodies are interspecies chimeric and humanizedantibodies; antibody fusions; and heteromeric antibody complexes, suchas diabodies (bispecific antibodies), single-chain diabodies, andintrabodies (see, e.g., Marasco (ed.), Intracellular Antibodies:Research and Disease Applications, Springer-Verlag New York, Inc. (1998)(ISBN: 3540641513), the disclosure of which is incorporated herein byreference in its entirety).

“Inhibiting” the onset of a disorder shall mean either lessening thelikelihood of the disorder's onset, delaying the disorder's onset, orpreventing the onset of the disorder entirely. In the preferredembodiment, inhibiting the onset of a disorder means preventing itsonset entirely.

“Islets” shall mean a composition comprising pancreatic islets, which issynonymous with the islets of Langehaus, which is the cluster of cellsin which the endocrine cells (that produce, e.g., insulin) are grouped.

“Islet cells” shall mean a composition comprising pancreatic isletcells, i.e., cells from the islets of Langehaus, which is the cluster ofcells in which the endocrine cells (that produce, e.g., insulin) aregrouped.

“Mammalian cell” shall mean any mammalian cell. Mammalian cells include,without limitation, cells which are normal, abnormal and transformed,and are exemplified by neurons, epithelial cells, muscle cells, bloodcells, immune cells, stem cells, osteocytes, endothelial cells and blastcells.

“Nucleic acid” shall mean any nucleic acid molecule, including, withoutlimitation, DNA, RNA and hybrids thereof. The nucleic acid bases thatform nucleic acid molecules can be the bases A, C, G, T and U, as wellas derivatives thereof. Derivatives of these bases are well known in theart, and are exemplified in PCR Systems, Reagents and Consumables(Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc.,Branchburg, N.J., USA).

“Operably affixed”, with respect to the second polypeptide of thisinvention, shall mean affixed (e.g., via peptide bond) in a mannerpermitting the ILT3 moiety thereof to inhibit the proliferation of CD4+T cells. In one embodiment, a polypeptide linker of 10, 11, 12, 13, 14,15 or 16 amino acid residues in length is used to join the ILT3 and Fcmoieties.

“Pharmaceutically acceptable carriers” are well known to those skilledin the art and include, but are not limited to, 0.01-0.1 M andpreferably 0.05 M phosphate buffer or 0.8% saline. Additionally, suchpharmaceutically acceptable carriers can be aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Porphyrin orLipofectin may also be used as a delivery agent. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions and suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's and fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers such as those based onRinger's dextrose, and the like. Preservatives and other additives mayalso be present, such as, for example, antimicrobials, antioxidants,chelating agents, inert gases, and the like.

“Polypeptide” and “protein” are used interchangeably herein, and eachmeans a polymer of amino acid residues. The amino acid residues can benaturally occurring or chemical analogues thereof. Polypeptides andproteins can also include modifications such as glycosylation, lipidattachment, sulfation, hydroxylation, and ADP-ribosylation. A variety ofmethods for labeling polypeptides and substituents or labels useful forsuch purposes are well known in the art, and include radioactiveisotopes such as 125I, 32 P, 35S, and 3H, ligands which bind to labeledantiligands (e.g., antibodies), fluorophores, chemiluminescent agents,enzymes, and antiligands which can serve as specific binding pairmembers for a labeled ligand. The choice of label depends on thesensitivity required, ease of conjugation with the primer, stabilityrequirements, and available instrumentation. Methods for labelingpolypeptides are well known in the art. See, e.g., Ausubel et al., 1992,hereby incorporated by reference.

“Prophylactically effective amount” means an amount sufficient toinhibit the onset of a disorder or a complication associated with adisorder in a subject. “Subject” shall mean any organism including,without limitation, a mammal such as a mouse, a rat, a dog, a guineapig, a ferret, a rabbit and a primate. In the preferred embodiment, thesubject is a human being.

“Therapeutically effective amount” means any amount of an agent which,when administered to a subject afflicted with a disorder against whichthe agent is effective, causes the subject to be treated.

“Transplant rejection” shall mean the adverse response by the immunesystem of a subject who has received a transplant (e.g., of an organ ortissue). Transplanted organs in this context include, for example,heart, kidney, skin, lung, liver, eye and bone. Transplanted tissue inthis context includes, for example, vascular tissue.

“Treating” a subject afflicted with a disorder shall mean causing thesubject to experience a reduction, delayed progression, regression orremission of the disorder and/or its symptoms. In one embodiment,recurrence of the disorder and/or its symptoms is prevented. In thepreferred embodiment, the subject is cured of the disorder and/or itssymptoms.

This invention provides a first polypeptide comprising all or a portionof the extracellular domain of ILT3, wherein the polypeptide iswater-soluble and does not comprise the Fc portion of an immunoglobulin.

In one embodiment, the polypeptide is isolated. In a further embodiment,the polypeptide comprises the extracellular domain of ILT3. In yet afurther embodiment, the polypeptide consists of the extracellular domainof ILT3. Preferably, the ILT3 is human ILT3. In one embodiment, theportion of ILT3 is the IgG1-like domain 1, the IgG1-like domain 2 or theN-terminal 250, 240, 230, 220, 210, 200, 190, 180, 170, 160 or 150 aminoacid residues of ILT3. In another embodiment, the portion of the ILT3 iscapable of inhibiting T cell proliferation or inducing differentiationof a T cell into a regulatory T cell. Assays to detect T cellproliferation and differentiation into regulatory T cells are well knownin the art and include those described below. Such polypeptides areuseful for preventing, inhibiting, reducing or suppressing immuneresponses mediated by the activation of T cells.

Also contemplated are polypeptides of the invention which contain minorvariations provided that the variations in the amino acid sequencemaintain at least 75%, more preferably at least 80%, 90%, 95%, and mostpreferably 99% sequence identity and the molecule retains bioactivity(e.g., inhibition of T cell proliferation, differentiation of T cellsinto regulatory T cells, suppression of immune responses mediated byactivated T cells). In particular, conservative amino acid replacementsare contemplated. Conservative replacements are those that take placewithin a family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into families: (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3)non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar=glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. More preferredfamilies are: serine and threonine are aliphatic-hydroxy family;asparagine and glutamine are an amide-containing family; alanine,valine, leucine and isoleucine are an aliphatic family; andphenylalanine, tryptophan, and tyrosine are an aromatic family.

This invention also provides a second polypeptide comprising (i) all ora portion of the extracellular domain of ILT3 operably affixed to (ii)the Fc portion of an immunoglobulin, wherein the Fc portion of theimmunoglobulin comprises a function-enhancing mutation, and wherein thepolypeptide is water-soluble. The Fc portion may also be substitutedwith any other peptide that promotes dimerization or oligomerization.For example, the peptide may comprise cysteine residues that formdisulfide bonds or other residues that promote covalent or nonconvalentinteractions between the peptides such that the peptides mediatedimerization or oligomerization. Suitable peptides include leucinezippers (e.g., those derived from the yeast GCN4 or a modified versionthereof. Other exemplary oligomerization domains are described in, e.g.,wo 00/69907, wo 99/62953, WO 98/56906, WO 98/18943, and wo 96/37621.

In one embodiment, the polypeptide is isolated. In a further embodiment,the polypeptide comprises the extracellular domain of ILT3. Preferably,the ILT3 is human ILT3. In a further embodiment, the Fc portion of theimmunoglobulin is the Fc portion of IgG 1. Preferably, the IgG1 is humanIgG1. In a further embodiment, the function-enhancing mutation in the Fcportion of the immunoglobulin inhibits the binding of the Fc portion ofan immunoglobulin to an Fc receptor. In one example, thefunction-enhancing mutation in the Fc portion of the immunoglobulin isan Asn-->Gln point mutation at amino acid residue 77 of the Fc portionof human IgG1.

This invention further provides a third polypeptide comprising (i) allor a portion of the extracellular domain of ILT3 operably affixed to(ii) a transmembrane domain. In one embodiment the transmembrane domaincorresponds to or is derived from the transmembrane domain of human ILT3(e.g.1 amino acid residues 259 to 280 of the sequence of GenBankAccession No. U82979). In another embodiment/the transmembrane domain isderived from a protein other than human ILT3 wherein the proteincomprises a transmembrane domain. Nucleic acids encoding thesepolypeptides/expression vectors and host cells comprising the nucleicacids and methods for producing these polypeptides are also provided.

Also contemplated are polypeptides of the invention which contain minorvariations provided that the variations in the amino acid sequencemaintain at least 75% 1 more preferably at least 80%1 90%1 95%1 and mostpreferably 99% sequence identity and the molecule retains bioactivity(e.g.1 inhibition of T cell proliferation/differentiation of T cellsinto regulatory T cellsl suppression of immune responses mediated byactivated T cells). In particular/conservative amino acid replacementsare contemplated. Conservative replacements are those that take placewithin a family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into families: (1)acidic=aspartate/glutamate; (2) basic=lysine1 arginine/histidine; (3)non-polar=alanine1 valine1 leucine1 isoleucine/proline1phenylalanine/methionine/tryptophan; and (4) uncharged polar=glycine1asparagine/glutamine/cysteine1 serine/threonine1 tyrosine. Morepreferred families are: serine and threonine are aliphatic-hydroxyfamily; asparagine and glutamine are an amide-containing family;alanine/valine1 leucine and isoleucine are an aliphatic family; andphenylalanine, tryptophan, and tyrosine are an aromatic family.

This invention provides a first isolated nucleic acid which encodes apolypeptide comprising all or a portion of the extracellular domain ofILT3, wherein the polypeptide is water-soluble and does not comprise theFc portion of an immunoglobulin. This invention includes nucleic acidsencoding polypeptides of the invention containing a conservativemutation as described above.

This invention further provides a second isolated nucleic acid whichencodes a polypeptide comprising (i) all or a portion of theextracellular domain of ILT3 operably affixed to (ii) the Fc portion ofan immunoglobulin, wherein the Fc portion of the immunoglobulincomprises a function-enhancing mutation, and wherein the polypeptide iswater-soluble.

In one embodiment of the instant nucleic acids, the nucleic acids areDNA (e.g., cDNA). In a further embodiment, the nucleic acids are RNA.

This invention provides a first expression vector comprising a nucleicacid sequence encoding a polypeptide comprising all or a portion of theextracellular domain of ILT3, wherein the polypeptide is water-solubleand does not comprise the Fc portion of an immunoglobulin. Thisinvention further provides a second expression vector comprising anucleic acid sequence encoding a polypeptide comprising (i) all or aportion of the extracellular domain of ILT3 operably affixed to (ii) theFc portion of an immunoglobulin, wherein the Fc portion of theimmunoglobulin comprises a function-enhancing mutation, and wherein thepolypeptide is water-soluble.

This invention provides a first host vector system which comprises thefirst expression vector and a suitable host cell.

This invention further provides a second host vector system whichcomprises the second expression vector and a suitable host cell.

The polypeptides of the invention may be expressed using any suitablevector. Typically, the vectors are derived from virus, plasmid,prokaryotic or eukaryotic chromosomal elements, or some combinationthereof, and may optionally include at least one origin of replication,at least one site for insertion of heterologous nucleic acid, and atleast one selectable marker. The invention also contemplates expressingthe polypeptides of the invention using artificial chromosomes, e.g.,bacterial artificial chromosomes (BACs), yeast artificial chromosomes(YACs), mammalian artificial chromosomes (MACs), and human artificialchromosomes (HACs), e.g., when it is necessary to propagate nucleicacids larger than can readily be accommodated in viral or plasmidvectors.

The vectors will also often include elements that permit in vitrotranscription of RNA from the inserted heterologous nucleic acid. Suchvectors typically include a phage promoter, such as that from T7, T3, orSP6, flanking the nucleic acid insert. Expression vectors often includea variety of other genetic elements operatively linked to theprotein-encoding heterologous nucleic acid insert, typically geneticelements that drive and regulate transcription, such as promoters andenhancer elements, those that facilitate RNA processing, such astranscription termination, splicing signals and/or polyadenylationsignals, and those that facilitate translation, such as ribosomalconsensus sequences. Other transcription control sequences include,e.g., operators, silencers, and the like. Use of such expression controlelements, including those that confer constitutive or inducibleexpression, and developmental or tissue-regulated expression arewell-known in the art.

Expression vectors can be designed to fuse the expressed polypeptide tosmall protein tags that facilitate purification and/or visualization.Many such tags are known and available. Expression vectors can also bedesigned to fuse proteins encoded by the heterologous nucleic acidinsert to polypeptides larger than purification and/or identificationtags. Useful protein fusions include those that permit display of theencoded protein on the surface of a phage or cell, fusions tointrinsically fluorescent proteins, such as luciferase or those thathave a green fluorescent protein (GFP)-like chromophore, and fusions foruse in two hybrid selection systems.

For long-term, high-yield recombinant production of the proteins,protein fusions, and protein fragments described herein, stableexpression is preferred. Stable expression is readily achieved byintegration into the host cell genome of vectors (preferably havingselectable markers), followed by selection for integrants.

The polypeptides of the invention may be expressed in any appropriatehost cell. The host cell can be prokaryotic (bacteria) or eukaryotic(e.g., yeast, insect, plant and animal cells). A host cell strain may bechosen for its ability to carry out desired post-translationalmodifications of the expressed protein. Such post-translationalmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation,hydroxylation, sulfation, lipidation, and acylation.

Exemplary prokaryotic host cells are E. coli, Caulobacter crescentus,Streptomyces species, and Salmonella typhimurium cells. Exemplary yeasthost cells are Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, and Pichia methanolica. Exemplary insect host cells arethose from Spodoptera frugiperda (e.g., Sf9 and Sf21 cell lines, andEXPRESSF™ cells (Protein Sciences Corp., Meriden, Conn., USA)),Drosophila S2 cells, and Trichoplusia ni HIGH FIVE® Cells (Invitrogen,Carlsbad, Calif., USA). Exemplary mammalian host cells are COS1 and COS7cells, NSO cells, Chinese hamster ovary (CHO) cells, NIH 3T3 cells, 293cells, HEPG2 cells, HeLa cells, L cells, MDCK, HEK293, WI38, murine EScell lines d(e.g., from strains 129/SV, C57/BL6, DBA-1, 129/SVJ), K562,Jurkat cells, BW5147 and any other commercially available human celllines. Other useful mammalian cell lines are well known and readilyavailable from the American Type Culture Collection (ATCC) (Manassas,Va., USA) and the National Institute of General Medical Sciences (NIGMS)Human Genetic Cell Repository at the Coriell Cell Repositories (Camden,N.J., USA).

In one embodiment of the instant host vector systems, the host cell is aeukaryotic, bacterial, insect or yeast cell. In a further embodiment,the host cell is a eukaryotic cell (e.g., a mammalian cell).

This invention provides a method for producing the first polypeptide,comprising (a) culturing the first host vector system under conditionspermitting polypeptide synthesis by the host vector system, and (b)recovering the polypeptide so produced.

This invention also provides a method for producing the secondpolypeptide, comprising (a) culturing the second host vector systemunder conditions permitting polypeptide synthesis by the host vectorsystem, and (b) recovering the polypeptide so produced.

This invention provides a first composition comprising (a) apharmaceutically acceptable carrier and (b) the first polypeptide.

This invention further provides a second composition comprising (a) apharmaceutically acceptable carrier and (b) the second polypeptide.

The polypeptides of the invention are administered to a subject viaparenteral injection (e.g., subcutaneous, intradermal, intraperitoneal,and intravenous). The polypeptides of the invention are administered,for example, once, a plurality of times, and/or over one or moreextended periods. They may be administered alone or I pharmaceuticalcompositions.

The polypeptides of the invention have immunosuppressive activity, whichact on T cells only upon their activation. Thus, these polypeptidesinduce antigen-specific tolerance. The polypeptides and compositions ofthe invention are useful for preventing, inhibiting, suppressing orreducing an immune response mediated by antigen-specific activation of Tcells. In one embodiment, the immune response is involved in transplantrejection. In another embodiment, the immune response is associated withan autoimmune disease, hypersensitivity or allergy. In yet anotherembodiment, the immune response is related to an inflammatory disorder.

This invention provides a method for inhibiting the onset of transplantrejection in a subject who has received, or is about to receive, atransplant, comprising administering to the subject a prophylacticallyeffective amount of the first, second or third polypeptide.

This invention further provides a method for treating transplantrejection in a subject who has received a transplant, comprisingadministering to the subject a therapeutically effective amount of thefirst, second or third polypeptide.

In certain embodiments of the methods for inhibiting the onset of andtreating transplant rejection, the transplant is an organ transplant. Inother embodiments, the transplant is a tissue transplant or involves thetransplantation of cells. Transplanted organs include, for example,pancreas heart, kidney, skin, lung, liver, eye, bone, and bone marrow.Transplanted tissue includes, for example, vascular tissue and islets.Transplanted cells include stem cells, e.g., umbilical cord stem cellsor adult stem cells, pancreatic islet cells, epithelial cells,endothelial cells, and liver cells. The transplant may also be aprosthetic device, e.g., stent. The transplant may be xenogeneic orallogeneic. In one embodiment, the subject is a mammal. Preferably, thesubject is a human.

In certain embodiments, the transplant is an islet transplant. Theislets can be transplanted by injection under the kidney capsules;however, other cell, tissue, and organ transplantation paradigms wellknown in the art can be used. It is contemplated that theimmunotherapeutic function of the present immunotolerance inductionregimen can be applied to transplantation of all or part of the pancreasas well as to the transplantation of pancreatic islets or islet cells.The donor can be a cadaver or a living donor. Furthermore, the donor canbe of the same species as the subject being treated or a differentspecies than the subject being treated. Thus, using the method of theinvention, transplantation can be performed across species (i.e.,xenogeneic transplantation or xenograft) and within the same species(i.e., allogeneic transplantation or allograft).

In one embodiment, the polypeptide is administered concurrently with asecond immunosuppressive agent, such as cyclosporine, OKT3 Antibody,rapamycin, Campath I, anti-CD69 antibody, thymoglobulin, andanti-thymocytic antibody. The polypeptide may also be administeredbefore or after administration of the second immunosuppressive agent. Inanother embodiment, the polypeptide is administered to the subject atthe time of transplantation and twice a week for two weeks as is routinefor transplants. In another embodiment, the polypeptide is administeredto the subject at the onset of or during rejection.

Symptoms associated with rejection of a transplant are well known in theart and include increased blood urea nitrogen (BUN) levels for kidney,increased glycemia for pancreas, lymphocyte infiltrates for heart, andincreased levels of enzymes such as aspartate aminotransferase (SGOT)and alanine aminotransferase (SGPT) for liver.

Histological indications of rejection of an islet transplant are wellknown in the art and include insulitis (infiltration of islets by CD8+T-cells), diffuse membrane immunostaining of CD40, and presence ofscattered apoptotic bodies in the islets.

This invention provides a method for treating a subject afflicted withan autoimmune disorder, comprising administering to the subject atherapeutically effective amount of the first, second or thirdpolypeptide.

The autoimmune disorder treated can be any such disorder, and includes,without limitation, rheumatoid arthritis, Crohn's disease, multiplesclerosis, autoimmune diabetes, systemic lupus erythematosus, lupusvulgaris, thyroiditis, Addison's Disease, hemolytic anemia,antiphospbolipid syndrome, dermatitis, allergic encephalomyelitis,glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, andautoimmune inflammatory eye disease. In one embodiment, the subject is amammal. Preferably, in the subject method, the subject is human. In oneembodiment, the polypeptide is administered to the subject during aflare-up of an autoimmune attack. The method may further compriseadministration of additional immunosuppressive drugs, e.g., cytotoxicagents, cyclosporine, methotrexate, azathioprine, and corticosteroids.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by a polypeptide of the present invention. These moleculescan be used to treat anaphylaxis, hypersensitivity to an antigenicmolecule, or blood group incompatibility.

This invention further provides a method for treating a subjectafflicted with an inflammatory disorder, comprising administering to thesubject a therapeutically effective amount of the first, second or thirdpolypeptide.

The inflammatory disorder treated can be any such disorder, andincludes, without limitation, (i) inflammatory diseases such as chronicinflammatory pathologies (including chronic inflammatory pathologiessuch as, but not limited to, sarcoidosis, chronic inflammatory boweldisease, ulcerative colitis, and Crohn's pathology); (ii) vascularinflammatory pathologies such as, but not limited to, disseminatedintravascular coagulation, atherosclerosis, Kawasaki's pathology andvasculitis syndromes (such as, but not limited to, polyarteritis nodosa,Wegener's granulomatosis, Henoch-Schonlein purpura, giant cell arthritisand microscopic vasculitis of the kidneys); (iii) chronic activehepatitis; (iv) Sjogren's syndrome; (v) spondyloarthropathies such asankylosing spondylitis, psoriatic arthritis and spondylitis,enteropathic arthritis and spondylitis, reactive arthritis and arthritisassociated with inflammatory bowel disease; and (vi) uveitis.Preferably, in the subject method, the subject is human. The method canalso be combined with administration of additional anti-inflammatoryagents. Anti-inflammatory agents include, but are not limited to, anyknown nonsteroidal anti-inflammatory agent such as, salicylic acidderivatives (aspirin), para-aminophenol derivatives(acetaminophen),indole and indene acetic acids (indomethacin), heteroaryl acetic acids(ketorolac), arylpropionic acids (ibuprofen), anthranilic acids(mefenamic acid), enolic acids (oxicams) and alkanones (nabumetone) andany known steroidal anti-inflammatory agent which includecorticosteriods and biologically active synthetic analogs with respectto their relative glucocorticoid (metabolic) and mineralocorticoid(electrolyte-regulating) activities. Additionally, other drugs used inthe therapy of inflammation include, but are not limited to, autocoidantagonists such as histamine, bradykinin receptor antagonists,leukotriene and prostaglandin receptor antagonists, and plateletactivating factor receptor antagonists.

This invention further provides a method for inducing anergy in a Tcell, thereby causing it to differentiate into a regulatory cell,comprising contacting the T cell with the first, second or thirdpolypeptide under conditions permitting priming of the T cell to occur,thereby inducing anergy in the T cell and causing it to differentiateinto a regulatory cell. Contacting of the T cell may be performed invivo, ex vivo or in vitro.

In one embodiment, the T cell is a CD4+ T cell, a CD3+ cell or a CD8+ Tcell, and the conditions permitting priming to occur comprise contactingthe T cell with an allogeneic stimulator (e.g., an allogeneic antigenpresenting cell (APC)) or an autologous APC that has been pulsed with anantigen. Exemplary antigen presenting cells include dendritic cells,monocytes, macrophages, endothelial cells and epithelial cells. In thepreferred embodiment, the allogeneic stimulator is an irradiated KG1cell.

This invention further provides a method for treating a subjectafflicted with an autoimmune disorder, comprising contacting, ex vivo,the first, second or third polypeptide with T cells obtained from thesubject, wherein the contacting is performed under conditions permittingpriming of the cells to occur, and intravenously administering theresulting cells to the subject, so as to treat the subject.

This invention also provides a method for treating transplant rejectionin a subject, comprising the steps of contacting, ex vivo, T cellsobtained from the subject with a polypeptide of the invention (e.g. thefirst, second or third polypeptide) under conditions permitting primingof the cells, and administering the resulting cells to the subject.

Methods of treating inflammatory disease or graft versus host disease ina subject by treating T cells obtained from the subject with apolypeptide of the invention ex vivo and then administering the treatedT cells to the subject are also contemplated.

In one embodiment, the T cell is a CD4+ T cell, a CD3+ T cell or a CD8+T cell and the conditions permitting priming to occur comprisecontacting the T cell with an allogeneic stimulator (e.g., an allogeneicantigen presenting cell (APC)) or an autologous APC that has been pulsedwith an antigen. Exemplary antigen presenting cells include dendriticcells, monocytes, macrophages, endothelial and epithelial cells. In thepreferred embodiment, the allogeneic stimulator is an irradiated KG1cell.

Determining an effective amount of the instant polypeptides for use inthe instant invention can be done based on animal data using routinecomputational methods. In one embodiment, the effective amount,administered intravenously, is between about 0.5 mg/kg and about 50mg/kg of polypeptide. In another embodiment, the effective amount,administered intravenously, is between about 1 mg/kg and about 20 mg/kgof polypeptide. In the preferred embodiment, the effective amount,administered intravenously, is about 3, 5 or 10 mg/kg of polypeptide.

In one embodiment of the instant methods, the polypeptide isadministered in a single dose. In another embodiment, the polypeptide isadministered in multiple doses.

Determination of a preferred pharmaceutical formulation and atherapeutically efficient dose regiment for a given application iswithin the skill of the art taking into consideration, for example, thecondition and weight of the patient, the extent of desired treatment andthe tolerance of the patient for the treatment.

Administration of the polypeptides or compositions of this invention,including isolated and purified forms, may be accomplished using any ofthe conventionally accepted modes of administration of agents which areused to prevent or treat transplantation rejection or to treatautoimmune or inflammatory disorders.

The pharmaceutical compositions of this invention may be in a variety offorms, which may be selected according to the preferred modes ofadministration. These include, for example, solid, semi-solid and liquiddosage forms such as tablets, pills, powders, liquid solutions orsuspensions, suppositories, and injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application. Modes of administration may include oral,parenteral, subcutaneous, intravenous, intralesional or topicaladministration.

The compositions of this invention may, for example, be placed intosterile, isotonic formulations with or without cofactors which stimulateuptake or stability. The formulation is preferably liquid, or may belyophilized powder. For example, the compositions or polypeptides of theinvention may be diluted with a formulation buffer comprising 5.0 mg/mlcitric acid monohydrate, 2.7 mg/ml trisodium citrate, 41 mg/ml mannitol,1 mg/ml glycine and 1 mg/ml polysorbate 20. This solution can belyophilized, stored under refrigeration and reconstituted prior toadministration with sterile Water-For-Injection (USP).

The compositions of the present invention can also be formulated so asto provide slow or controlled-release of the active ingredient thereinusing, e.g., hydropropylmethyl cellulose in varying proportions toprovide the desired release profile, other polymer matrices, gels,permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes and/or microspheres.

In general, a controlled-release preparation is a composition capable ofreleasing the active ingredient at the required rate to maintainconstant pharmacological activity for a desirable period of time. Suchdosage forms can provide a supply of a drug to the body during apredetermined period of time and thus maintain drug levels in thetherapeutic range for longer periods of time than other non-controlledformulations.

Also provided are methods of delivering membrane-bound polypeptides ofthe invention using lipid bilayers or by administration of cellsmanipulated to express the polypeptides of the invention (e.g., cellstransfected with a nucleic acid encoding a polypeptide of the invention)(M. Davis et al., JCB 166:579-590 (2004)).

Nucleic acids encoding a polypeptide of the invention may beadministered using gene therapy methods. Retrovirus vectors andadeno-associated virus (AAV) vectors are preferred vectors according tothe invention for transferring nucleic acids encoding the polypeptidesof the invention into cells in vivo, particularly into human cells.These vectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. The development of specialized cell lines (“packagingcells”) that produce replication-defective retroviruses are especiallypreferred for gene therapy applications (see, e.g., Miller, A. D. Blood76:271(1990)). Recombinant retrovirus may be constructed in which partof the retroviral coding sequence (gag, pol, env) has been replaced bynucleic acid encoding one of the subject receptors rendering theretrovirus replication defective. The replication defective retrovirusis then packaged into virions which can be used to infect a target cellthrough the use of a helper virus by standard techniques. Protocols forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses may be found, e.g., in Current Protocols inMolecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Representative examples of retroviruses include pLJ, pZIP, pWEand pEM which are well known to those skilled in the art. Representativeexamples of packaging virus lines for preparing both ecotropic andamphotropic retroviral systems include psi.Crip1 psi.Crel psi 2 andpsi.Am. Retroviruses have been widely used to introduce a variety ofgenes into many different cell types in vitro and/or in vivo. Moreover1it is useful to limit the infection spectrum of retroviruses andretroviral-based vectors by modifying the viral packaging proteins onthe surface of the viral particle (see1 for example PCT publicationsW093/25234 and W094/06920; Roux et al. PNAS 86:9079-9083(1989); Julan etal. J. Gen Virol 73:3251-3255(1992); and Goud et al. Virology163:251-254(1983)); Neda et al. J. Biol Chern 266:14143-14146(1991)).

This invention further provides an article of manufacture comprising (a)a packaging material having therein the first polypeptide/and (b) alabel indicating a use for the polypeptide for (i) treating orinhibiting the onset of transplant rejection in a subject (ii) treatingan autoimmune disorder in a subject, or (iii) treating an inflammatorydisorder in a subject.

Finally, this invention provides an article of manufacture comprising(a) a packaging material having therein the second polypeptide, and (b)a label indicating a use for the polypeptide for (i) treating orinhibiting the onset of transplant rejection in a subject, (ii) treatingan autoimmune disorder in a subject, or (iii) treating an inflammatorydisorder in a subject.

This invention provides a method for inhibiting the onset of, ortreating, transplant rejection in a subject who has received, or isabout to receive, a pancreatic islet cell transplant by administeringthe polypeptide to the subject.

This invention provides a method for inhibiting the onset of, ortreating, autoimmune diabetes by administering the polypeptide to thesubject.

This invention provides a method for inhibiting the onset of, ortreating, Graft Versus Host Disease (GVHD) by administering thepolypeptide to the subject.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS

The most extensively studied “protein therapeutic drugs” are (1)monoclonal antibodies, (2) cytokine-fusion proteins and (3) chimericcell adhesion molecules that prevent T cell activation and/orproliferation.

Soluble ILT3

Soluble ILT3 (“siLT3”) (encompassing the first and second instantpolypeptides) belongs to the family of chimeric proteins that modulatethe immune system. siLT3 is an attractive candidate for therapeutic usebased on its potent in vitro immune modulating activities and orthologue(homolog) of ILT3 studies shown in animal models of acute and chronicinflammation, autoimmunity. Soluble ILT3 induces immune tolerance byinhibition of T cell activation that contributes to graft rejection.

Examples of soluble chimeric proteins being investigated as therapeuticagents in clinical trials are CTLA-4, CD86, PD-1 and PD-L1 fusionproteins. Protein therapeutics are administered through a parenteralroute, e.g., subcutaneous, intravenous, intradermal, or intraperitonealroutes. Some have been proven effective against specific autoimmunedisease. However, these molecules behave both stimulatory and inhibitorymanner depending on their counter-receptors. Furthermore, theirexpression is ubiquitous such that CTLA-4 and its counter-receptor areexpressed on the same T cells. Moreover, the broad expression pattern oftheir counter-receptors on B cell, DCs, ECs, macrophages, fibroblasts,muscle cells and trophoblast cells render them less-specific for theinhibition of T cell proliferation.

In this respect, ILT3 is unique. Its expression is limited to dendritic(professional) and endothelial (semi-professional) antigen presentingcells, two cell types playing an important role in modulating the immuneresponses. They can be either immunogenic or tolerogenic. However, theexpression of ILT3 on these two cell types renders them tolerogenicleading to induction of anergy. ILT3 can be induced with inhibitorycytokines (IL1O, IFNa) and Vitamin D3 and render DC and EC tolerogenic.However, one can not ignore the possible other, as yet undiscoveredeffects of cytokine treatments on cells. Thus, having siLT3 directlyinteract with T cells, rendering them anergic, will be more effective indown-regulating the immune system.

This invention offers several advantages. First, the second polypeptideof this invention has an extended circulating half-life and provideslong-term protection, acting as a long-lasting “chimeric” protein drug.The prolonged half-life permits lower dosing, thereby reducing toxicity.Second, soluble polypeptides (i.e. the extracellular domain of ILT3) andlongevity increasing polypeptides (i.e., Fc portion of Ig) useful inthis invention can readily be isolated using routine methods. siLT3 canbe administered subcutaneously, intradermally, intraperitoneally, orintravenously.

Example 1

Construction of Cells Expressing ILT3 or ILT3delta

KG1 cells over-expressing human ILT3 (KG1.ILT3 cells) were generated aspreviously described (CC Chang et al., Nat. Immunol. 3:237-243, 2002).Deletion of the cytoplasmic region of ILT3 was accomplished by PCRamplification using the following primers:sense-5′-CCATGATATCAGGAGACGCCATGATCCCCA-3′ (SEQ ID NO: 1) andantisense-5′-ATGTAGCGGCCGCGTTTTCTCCCTGGACGTCA-3′ (SEQ ID NO: 2) and aplasmid containing a full-length cDNA of ILT3 (pcDNA4-ILT3) was used astemplate. PCR conditions were as follows: 5 min 94° C. 30 cycles (30 sec94° C., 1 min 68° C., 1 min 72° C. 7 min 72° C. The PCR product waspurified using a PCR purification kit {Qiagen) and subcloned into theEcoRV and Noti sites of the expression vector pcDNA4/TO/myc-His in framewith a c-myc-His epitope (Invitrogen). The resulting ILT3 deletionmutant, ILT3delta (which contains residues 1 (Met) to 328 (Asn) of humanILT3), encodes a protein that contains the putative leader peptide, theextracellular and transmembrane domains and a stretch of 48 amino acidsof the cytoplasmic domain of ILT3 followed by a c-terminal myc-His tag.The ILT3delta insert was subcloned into the Bglii site of retroviralvector MIG (MSCV-IRES-GFP) and the resulting construct was confirmed bysequencing. The ILT3delta was over-expressed in KG1 cells by retroviraltransduction (Change et al., supra). Transfectants were sorted for GFPexpression by flow cytometry.

Generation of Soluble ILT3-Fc Chimeric Protein

cDNA fragment coding for the extracellular domain of human ILT3 wasfused to the Fc portion of the human IgG1 heavy chain. To abolishbinding to the Fc receptor, a mutation was introduced in the N-linkedglycosylation site, N77 (Asn>G1n) of the Fc domain. Expression vectorpcDNA3 (Invitrogen) containing the ILT3-Fc fusion gene (FIG. 1) wastransfected into CHO-S cells. Homogenous cell populations were obtainedby limiting dilution and clones with high expression of ILT3 (asdetermined by RT-PCR and Western Blot analysis) were selected. ILT3-Fcfusion proteins were purified from the supernatant of the selectedclones using a recombinant protein A FF column and analyzed by Westernblotting using an anti-human Fc-specific antibody.

Example 2

Membrane ILT3 Induces CD4+ TH Cell Anergy and Inhibits the Generation ofCDB+ Cytotoxic Cells

The cytoplasmic region of ILT3 contains ITIM motifs that recruitinhibitory phosphatases, which can negatively regulate cell activation.The extracellular portion contains two Ig domains, one or both of whichare likely to contribute to the ILT3 ligand binding sites involved inthe interaction of APC with T lymphocytes. By overexpressing thecytoplasmic tail deletion mutant ILT3delta in KG1 cells, we generatedthe KG1.ILT3delta cell line, which we then used to explore the activityof membrane ILT3 (miLT3) (FIG. 4A).

KG1.ILT3 and KG1.ILT3delta expressed similar amounts of ILT3 protein onthe cell surface as shown by flow cytometry analysis using mAb to ILT3(FIG. 4B-FIG. 4C). Western blot analysis using anti-myc antibody, whichbinds to the c-terminal myc tag of the recombinant proteins,demonstrated that the molecular weight of the ILT3delta protein was 38kDa while that of full length ILT3 was 50kDa (FIG. 4D).

ILT3 associates with SHP-1 phosphatase and this association is increasedby receptor crosslinking with specific antibodies or with pervanadatetreatment. SHP-1 recruitment upon phosphorylation of the cytoplasmicITIMs has been shown to mediate the negative signaling of ILT2, aclosely related member of the same family of Ig-like inhibitoryreceptors as ILT3. Immunoprecipitation experiments using the anti-SHP-1antibodies and western blot analysis using an anti-myc antibody showedconstitutive interaction of the full-length ILT3 molecule and SHP-1. Asexpected, no interaction of SHP-1 with ILT3delta was observed (FIG. 4D).

Antibodies against HLA-DR can trigger both Ca²+mobilization and specificprotein phosphorylation. Co-crosslinking of the anti-HLA-DR antibodywith anti-ILT3 antibody results in substantial inhibition or more rapidextinction of the activation signal (Cella et al., J. Exp. Med.,185:1743-1751, 1997). To establish whether lack of SHP-1 recruitment byILT3delta was accompanied by a lack of inhibition of protein tyrosinephosphorylation, KG1.ILT3 and KG1.ILT3delta cells were ligated withanti-HLA-DR mAb or with anti-HLA-DR mAb and anti-ILT3 mAb in thepresence of a crosslinking antibody. Total cell extracts were analyzedby western blot with anti-phoshotyrosine mAb and reprobed withanti-β-actin mAb for control of equal loading. The results showed thatcrosslinking HLA-DR and ILT3 on KG1.ILT3 cells inhibits tyrosinephosphorylation, however, it has little or no effect on tyrosinephosphorylation in KG1.ILT3delta mutants (FIG. 4E).

Comparison of the capacity of KG1, KG1.ILT3 and KG1.ILT3delta to elicitT cell proliferation in primary and secondary mixed leukocyte culture(MLC) showed that KG1.ILT3 and KG1.ILT3delta elicited much lessproliferation of unprimed (FIG. 5A) or KG1-primed T cells (FIG. 5B) thanKG1 cells.

For the proliferation assays, responding T cells (5×10⁴/well) weretested for reactivity to irradiated KG1, KG1.ILT3, KG1.ILT3delta, orallogeneic CD2-depleted APC (2.5×10⁴/well). After 5 days (for naive Tcells) or 2 days (for primed T cells) of incubation, the cultures werepulsed with [³H]-thymidine and harvested 18 hours later. [³H]-thymidineincorporation was determined by scintillation spectrometry in an LKB1250 Betaplate counter. Mean counts per minute (c.p.m.) of triplicatecultures and the standard deviation (sd) to the mean were calculated.

Addition of anti-ILT3 monoclonal antibody (5 pg/ml) or IL-2 (10 U/ml) tothe blastogenesis assays restored T cell proliferation in response toKG1.ILT3 and KG1.ILT3delta (FIG. 5B). These experiments indicate thatmiLT3 protein is sufficient for inducing an inhibitory signal inactivated T cells and that deletion of the cytoplasmic region of ILT3does not abrogate its T cell anergizing activity.

To study the effect of ILT3 on the generation of cytotoxic T cells,CD3+CD25− T cells were primed with KG1, KG1.ILT3 or KG1.ILT3delta. After7 days, CD8+ T cells were isolated from each of the cell cultures andtested for their ability to kill KG1 cells. T cells primed with KG1.ILT3or KG1.ILT3delta showed significantly less cytotoxic activity (6%) thanT cells primed with KG1 (24%) as determined by Annexin V/PropidiumIodine staining (FIG. 5C-5E) and produced less IFN-gamma. Thus, T cellinteraction with miLT3 inhibits the differentiation of cytotoxiceffector cells.

siLT3 Induces CD4+ TH Cell Anergy and Inhibits the Generation of CDB+Cytotoxic Cells

siLT3 expression was assayed by Western blotting. Briefly, cell extractsat equal concentration were immunoprecipitated from cleared extractusing mouse anti-ILT3 mAb (ZM 3.8) and subjected to SDS-PAGE. Proteinswere then electrotransferred onto polyvinylidene difluoride (PVDF)membrane, and were incubated with anti-human Fc polyclonal antibody.Immunoblots were developed by ECL and acquired by thephosphor/fluorescence imager. Apparent molecular weight of soluble ILT3is about 90 and 50 kDA in non-reducing and reducing conditions,respectively.

Responding CD3+CD25− or CD4+CD25− cells (1×105 cells/well) werestimulated with irradiated KG-1 cells (30 min) (0.5×105 cells/well) inthe presence or absence of siLT3-Fc fusion protein in 96-well, roundbottom microtiter plates. At 5 days later, cell proliferation wasdetermined by pulsing with [³H] thymidine (0.5 pCi/well) overnight andradioactivity was counted on a beta reader. Addition of 50 pg/ml siLT3inhibited cell proliferation by >90% (FIG. 3A-FIG. 3B and FIG. 6).Similarly, when 50 pg/ml siLT3 was added to secondary MLC at the time ofrestimulation there was >70% inhibition of the secondary response (FIG.6).

Analysis of the capacity of siLT3 to inhibit generation of cytotoxic Tcells showed that CD8+ T cells primed in 7-day cultures with KG1 cells,in the presence of siLT3 (50 pg/ml) were devoid of killing capacity(FIG. 7). Furthermore, CD8+ T cells from these cultures did not produceIFN-gamma as demonstrated in ELISPOT assays (FIG. 8). Taken togetherthese data indicate that siLT3, similar to miLT3, induces TH anergy andblocks the generation of cytotoxic T cells.

Example 3

miLT3 and siLT3 Induce the Generation of Regulatory/Suppressor T Cells

The finding that siLT3 inhibits T cell proliferation in response toallogeneic stimulating cells suggested the possibility that this proteininduces anergy in primed T cells triggering their differentiation intoregulatory cells.

To explore this hypothesis, umprimed CD4+ or CD8+ T cells were incubatedwith allogeneic stimulators (irradiated KG1 cells) in the presence orabsence of siLT3 (50 pg/ml). After 7 days, T cells were harvested fromthe cultures and tested for: (a) expression of FOXP3 (a Tsuppressor/regulatory cell marker) and (b) capacity to inhibit MLCreactions.

CD4+ and CD8+ T cells primed in the presence of siLT3 for 7 daysexpressed FOXP3 and suppressed KG1-triggered proliferation of naive CD4+T cells in MLC. Control T cells primed for 7 days in cultures with siLT3or with siLT3 but without allogeneic stimulating cells did not acquireregulatory function. CD8+ T cells primed in the presence of siLT3induced dose-dependent inhibition of T cell proliferation from 50% at a0.25:1 ratio of primed CD8+ Ts to responding CD4+ TH cell ratio to 90%at a 1:1 ratio (FIG. 9A). CD8+ T cells primed in cultures without siLT3induced 20% inhibition at the highest concentration and virtually noinhibition at lower doses.

The capacity of miLT3 to induce the generation of CD8+ T suppressorcells was also tested. CD3+CD25− T cells were primed for 7 days eitherwith KG1 or KG1-ILT3delta cells and then CD8+ T cells were isolated andtested for their capacity to inhibit the response of unprimed,autologous CD4+ T cells to KG1. CD8+ T cells primed to KG1-ILT3 induceddose-dependent inhibition of T cell response to KG1 while CD8+ T cellsprimed to KG1 showed inhibitory activity (35%) only at a 1:1 ratio (FIG.9B).

Therefore siLT3 as well as miLT3 induces the differentiation of CD8+ Tsuppressor cells in primary MLC. Since FOXP3 is a characteristic markerfor CD4+ and CD8+ regulatory T cells, its expression in CD8+ T cellsprimed for 7 days to allogeneic APC in the presence or absence of siLT3(50 g/ml) was determined. CD8+ T cells from cultures stimulated eitherwith KG1 or with KG1.ILT3delta were also tested for FOXP3 expression.Western Blot analysis using mAb to FOXP3 (FIG. 9C) showed that bothsiLT3 and miLT3 induced CD8+ T cells with suppressor activity and highexpression of FOXP3. Taken together these data indicate that both siLT3and miLT3 induce the differentiation of CD8+ Ts with potent inhibitoryactivity.

Example 4

siLT3 induces the Generation of CDB+ T suppressor cells by Interactionwith CD4+ T cells CDB+ T suppressor cells generated by multiple in vitrostimulation with allogeneic APC were previously shown to act directly onAPC, inducing the down regulation of costimulatory molecules and theupregulation of ILT3 and ILT4. To determine whether T suppressor cellsgenerated by allostimulation in the presence of siLT3 have a similareffect on APC, we tested CD8+ T cells primed under such conditions fortheir ability to modulate CD86 and ILT3 expression on DC from the donorused for priming and on control DC from an individual sharing no HLAclass I antigens with the original stimulator.

CD8+ T cells isolated from the culture containing siLT3 were able todramatically upregulate the expression of ILT3 on DC from the specificstimulator but not on control APC. This alloantigen-specificupregulation of the inhibitory receptor ILT3 occurred in conjunctionwith the downregulation of CD86 (FIG. 9D-FIG. 9E).

To determine if the generation of CD8+ T suppressor cells is due todirect interaction of ILT3 with CD8+ T cells or if it results indirectlyfrom interaction between ILT3 and CD4+ T cells, FITC-labeled siLT3protein was used to stain T cells in primary MLC. CD4+ T cells, but notCD8+ T cells were stained by siLT3-FITC indicating that directinteraction of ILT3 and CD8+ cells is not involved in the generation ofCD8+ T suppressor cells (FIG. 10A-FIG. 10B).

The discovery that siLT3 has potent immunosuppressive activity and thatit acts on T cells only upon activation has important clinicalimplication indicates that siLT3 is useful for inducing antigen specifictolerance. siLT3 signaling in T lymphocytes via its ligand may interferewith the generation of effective immunity, promoting the generation of Tcells with suppressive function. Because siLT3 has no effect on resting,non-stimulated T cells it is likely to inhibit allograft rejectionmediated by T cells activated via direct or indirect pathways.Administration of siLT3 can also attenuate the proliferation of T cellsinvolved in aggressive autoimmune responses triggered by activated DCwhich cross present immunogenic self-peptides. Attenuation of activationefficiency may result in stalled immune responses which perpetuatequiescence.

These findings have important clinical implications because they opentwo new avenues for antigen-specific suppression of the immune responsein transplantation and autoimmune diseases.

The first avenue resides in treating transplant recipients at the timeof or after transplantation or at the onset of an acute rejectionepisode with siLT3. This molecule is expected to bind only to T cellsthat have been activated by the transplant's alloantigens and not toumprimed T cells, thus suppressing the immune response in anantigen-specific manner. Similarly, siLT3 administration during theflare-up of an autoimmune attack, e.g., in rheumatoid arthritis, Crohn'sdisease, multiple sclerosis, or onset of type I JDM, may prevent theevolution, e.g., progression of the disease.

The second avenue is to use cell therapy, by leukophoresing the patientand exposing the harvested cells to siLT3 for 18 h. In vitro activated Tcells, but not unprimed T cells, are expected to be converted intoregulatory cells which, when reinfused into the patient, should blockthe progression of the immune response.

Example 5

Islet Cell Transplantation in Humanized NOD/SCID Mice

Although many biological mechanisms are similar in rodents and humans,there are several structural and functional differences which can renderthe extrapolation of experimental results to clinical practicedifficult. Thus, humanized mice, defined as immunodeficient miceengrafted with human haematopoietic stem cells or PBMC, provide apowerful and productive tool for preclinical testing of newimmunomodulatory agents and study of human immune responses. This isparticularly true in the case of ILT3 which, like other members of theImmunoglobulin Gene Superfamily, has no ortholog in rodents.

To demonstrate that siLT3 prolongs islet allograft survival we used thehumanized Non-Obese Diabetic/Severe Combined Immunodeficiency(hu-NOD/SCID) mouse model developed by Gregori et al. NOD mice are usedas an animal model for type 1 diabetes. SCID mice present without theability to make T or B lymphocytes. As such, the mice cannot fightinfections and are also unable to reject tumors or transplants. NOD/SCIDmice (N=89) were rendered diabetic by a single injection ofstreptozotocin (STZ; Sigma-Aldrich) at a dose of 180mg/kg. STZ-toxicitycaused the death of 33% of the injected animals (29/89) within 72 hours.Blood glucose levels of the surviving mice were measured twice a weekusing Ascensia Elite XL Blood Glucose Meter system (Bayer AG). Diagnosisof diabetes was based on two consecutive glucose measurements >350mg/dl. All but 8 of the remaining 60 animals (87%) became diabetic 3-6days after STZ injection.

The diabetic animals were then transplanted with 1500 IEQs (isletequivalent) of human islets, via injection under the kidney capsule asdescribed in Davalli et al. (1996, Diabetes, 45:1161-1167; fullyincorporated herein by reference). Purified human pancreatic islets fromhuman were obtained from the National Islet Cell Resource CenterProgram. The islets were cultured in CMRL1066 culture mediumsupplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and100 mg/ml streptomycin at 37° C. in a 5% C02 , humidified atmosphere.All islet samples were >70% pure and 90% viable. Purity was determinedby the percentage of dithizone-positive particles, and viability wasdetermined by fluorescein diacetate and propidium iodine staining.

Following islet cell transplantation, mice that did not achieveeuglycemia (glucose level <100 mg/dl) were eliminated from the study onthe assumption that the grafted islets were not functional (Some animalsdied 48 hrs post-operation and others received islet cells that werenon-functioning and failed to recover from the STZ-induced diabetes).Out of the fifty-two animals, thirty-two were successfully transplanted,as they became euglycemic within 7 days.

7 to 10 days post-transplantation, mice that were restored to euglycemiawere humanized. That is, they received an intraperitoneal (i.p.)injection of 50×106 freshly isolated peripheral blood mononuclear cells(PBMC) from healthy human blood donors, isolated from fresh buffy coatspurchased from the New York Blood Center. These PBMC then develop intofunctioning T-cells, restoring immune function to the immunodeficientmice.

Ten days after PBMC injection, circulating human T cells (whichdeveloped from the injected PBMC) taken from heparizined retro-orbitalvenous samples were evaluated by flow cytometry. Animals that failed tobe reconstituted with human T cells were excluded from further analysisby prior design. To avoid variability between samples, both islets andPBMC were administered to mice from the siLT3 and human IgG group in apairwise fashion.

Immunomodulatory Effect of ILT3-Fc in Islet Cell Transplantation inDiabetic Hu-NOD/SCID Mice

Concurrently with the PBMC injection, the mice were begun on a treatmentregimen. The mice were assigned to one of three groups: 1) the treatmentgroup, which received a daily intraperitoneal injection of 250 pg siLT3,for ten consecutive days 2) the IgG control group, which received 250 pghuman IgG instead, and 3) the no-treatment control group, which receivedno treatment.

Blood glucose levels were measured twice a week before, during, andafter the treatment period in order to assess the health of the isletcell graft. Within the group of IgG-treated control animals (N=16)1eight rejected the graft within 3 to 7 weeks. Those mice again becamediabetic1 demonstrated by the increase in blood glucose above 350 mg/dl1as well as by histological studies (described below). Similar resultswere seen in the no-treatment control group. By contrast1 none of theILT3-Fc treated hu-NOD/SCID mice became diabetic over 91 days ofobservation (Table 1 and FIG. 11). Animals that developed graft versushost disease (hunched back1 lethargy/weight loss/and tachypnea) wereexcluded from the analysis of rescue from diabetes. Actuarial freedomfrom diabetes was 100% in the ILT3-Fc treatment group1 indicating thatsoluble ILT3 (siLT3) inhibited rejection of islet allografts in allhu-NOD/SCID recipients (p=O0.0001) (FIG. 11).

Using ILT3-Fc treatment1 we prevented rejection of islet grafts in 100%of hu-NOD/SCID recipients. To our knowledge this is the highest rate ofsuccessful transplantation of allogeneic human islets in a pre-clinicalmodel in which the efficacy of a biological agent was tested alone1without any complementary pharmaceutical immuno-suppression.

TABLE 1 Blood glucose levels in hu-NOD/SCID islet allograft recipientstreated with ILT3-Fc or control human IgG. Week Control group Treatmentgroup P-value Week n = 16 84.4 ± 31.1 n = 16 79.3 ± 10.4 N.S. #1 Week n= 16 80.1 ± 22.0 n = 16 77.3 ± 9.4  N.S. #2 Week n = 16 103.7 ± 55.4  n= 16 74.9 ± 9.0  P = 0.057 #3 Week n = 13 104.1 ± 30.2  n = 15 76.6 ±11.9 P = 0.008 #4 Week n = 12 197.3 ± 103.9 n = 13 73.6 ± 8.6  P = 0.002#5 Week n = 8  237.8 ± 122.7 n = 12 78.8 ± 10.3  P = 0.0008 #6 Week n =4  279.8 ± 91.9  n = 12 81.9 ± 22.2 P = 0.02  #7* *Due to attrition inthe control group, no statistical comparison can be performed after week7.

Example 6

In Vivo Generation of Regulatory and Suppressor T Cells in ILT3-FcTreated Animals.

We further demonstrated that the human PBMC injected into thehu-NOD/SCID mice differentiate into regulatory T cells in vivo, and thatthe administration of ILT3-FC to the host favored the differentiation ofPBMC into T cells with immuno-suppressive activity, e.g., CD8+suppressor T (Ts) cells. Four hu-NOD/SCID mice, prepared in the same wayas those described above, were transplanted with islets from a humandonor expressing HLA-A1, B8, DR3/A2, B44, and DR7. Two mice were treatedwith ILT3-Fc, and the other two were treated with IgG. One from eachpair was sacrificed on day 23 after human PBMC injection, when glycemiawas 240 mg/dl (signaling the onset of transplant rejection and relapseof diabetes) in the IgG-treated mouse and 72 mg/dl (normal glycemiclevels) in the ILT3-Fc treated mouse. The remaining two were sacrificedon day 47. At the time of sacrifice at day 47, the IgG-treated mouse hada diabetic blood glucose level of 3SO mg/dl and the ILT3-Fc-treatedmouse had a euglycemic blood glucose level of SO mg/dl.

Human CD8+ and CD4+ T cells were magnetically sorted from the spleen ofthe hu-NOD/SCID mouse recipients (using isolation kits by StemCellTechnologies). These sorted CD4+ or CD8+ T-cells were added atincreasing numbers (1-8×10⁴/well) to a fixed number (10⁴/well) ofunprimed autologous CD3+CD25− T-cells (used as responders) andstimulated for 6 days in mixed leukocyte culture (MLC) with irradiated,allogeneic PBMC sharing HLA-A, -B, and -DR antigens with the islettransplant donor. The cultures were harvested after 6 days, and[³H]thymidine incorporation was measured to assay T cell reactivity.

CD8+ T cells obtained from the ILT3-Fc treated mice sacrificed on bothdays 23 and 47 (after PBMC injection) suppressed T cell reactivity,confirming their identity as Ts cells (FIG. 13A-FIG. 13F). For example,CD8+ T cells isolated on day 47 from ILT3-Fc-treated mice suppressed thereactivity of responder T cells by 7S % when cultured at an 8:1 ratio ofregulatory to responder T cells (FIG. 13A-FIG. 13F, Mouse pair #2, leftgraph). At this ratio (of 8:1), CD4+ T cells isolated from these ILT3-Fctreated animals also had suppressive activity, and suppressed T cellactivation by 21% (FIG. 13A-FIG. 13F, Mouse pair #2, right graph). Bycontrast, T cells generated from injected human PBMC in the control mice(IgG-treated, lacking ILT3 treatment) did not become Ts cells. CD8+ Tcells isolated from the control mice (IgG-treated) lackedimmunosuppressive activity, and CD4+ T cells isolated from the controlanimals even had a mild immuno-activatory effect (mouse pairs #1 and #2,right graphs). Therefore, the administration of LIT3-Fc facilitatedimmunosuppression by facilitating the production of CD8+ Ts cells, aswell as CD4+ T cells with immunosuppressive activity.

To show that the presence of CD8+ Ts cells is associated with toleranceto the allogeneic islet transplants, we tested CD8+ and CD4+ T cellsisolated from the spleens of ILT3-Fc-treated euglycemic hu-NOD/SCID micesacrificed on day 91. Antigen-presenting cells (APCs) sharing HLA-classI and class II antigens with the islet graft were used for stimulating Tcells autologous to the cells tested for suppressive activity. Asillustrated in FIG. 13 (Row three-Tolerant Mouse), human CD8+ T cellsisolated from mice with long lasting tolerance (N=S) displayedsuppressive activity. CD4+ T cells from the same animals also showedweak suppressive activity (<20%). Therefore, the data demonstrate thatadministering ILT3-Fc to the host prevents islet allograft rejection,and does so by inducing the formation of regulatory T cells, e.g., CD8+Ts cells and CD4+ T cells with immunosuppressive activity.

Overall engraftment of human T cells into recipients' spleens wasunaffected by ILT3-Fc administration, and the distribution of CD4+ andCD8+ cells, as well as CD14+ and CD19+ cells, was similar between theILT3-Fc and human IgG-treated mice, as illustrated in FIG. 14A-FIG. 14B.These experiments served as valuable controls, and demonstrated thatadministration of ILT3-Fc into the host reduced allogeneic transplantrejection through inducing the differentiation of transplanted PBMC intoregulatory T cells, e.g., CD8+suppressor T (T8) cells, rather thanthrough a non-specific effect such as reduced overall engraftment ofPBMC.

The Effect of siLT3 Administration on Gene Expression in Engrafted HumanT Cells

To further characterize the phenotype and function of effector and Tscells present in the transplant recipient mice, we performed real timePCR studies to assay the expression of inflammatory cytokines by humanCD4+ and CD8+ cells sorted from the spleens of these mice sacrificed ondays 23 and 47 (after humanization). Total RNA was isolated with theRNAqueous-4PCR kit (Stratagene, La Jolla, Calif.). Complementary cDNAwas synthesized using the 1st Strand cDNA Synthesis Kit for RT-PCR(Roche Diagnostics, Basel, Switzerland). Real-time PCR was performedusing Taqman gene expression primer probes (Applied Biosystems, FosterCity, Calif.). Data were collected and analyzed with the 7300 SDS 1.3.1software (Applied Biosystems). The relative amount of gene expressionwas calculated by the formula: 2-C.ct, whereCt=[Ct(gene)Ct(glyceraldehyde-3-phosphate dehydrogenase)] and Ct is the“crossing threshold” value returned by the PCR instrument for every geneamplification. We found that treatment with ILT3-Fc inhibited thecapacity of both CD4+ and CD8+ T-cells to produce Th1-type (IFN-7 andIL-2) and Th2-type (IL-5 and IL-10) cytokines (FIG. 15A=FIG. 15B).

We also assayed the expression of T-cell activation markers CD28 andCD40L by human CD8+ T-cells colonizing the spleens using flow cytometry.Flow-cytometry studies were performed on a FACS Calibur instrument usingsix-parameter acquisition (BD Biosciences). The frequency of CD8+CD28+T-cells was significantly lower on weeks 4 and 7 (P =0.011 and 0.04 S)in ILT3-Fc-treated animals compared with paired controls as illustratedin FIG. 15C. Similarly, the frequency of CD8+CD40L+ was significantlylower (P=0.007 and 0.022) in ILT3-Fc-treated animals compared withpaired controls (see FIG. 15C). This down-regulation of CD28 expressionin CD8+ T cells from animals treated with ILT3-Fc corroborates our, andother investigators' previous, findings that CD8+ Ts cellscharacteristically have low expression of CD28. In addition, theCD40-CD40L costimulatory pathway is deemed to be crucial to theactivation and differentiation of T effector cells. Because CD40 isexpressed by pancreatic islet cells, a down-regulation of CD40L alsocorroborates the immunosuppressive action of ILT-3 administration.

These results demonstrate that primed CD8+ T-cells from ILT3-Fc-treatedanimals differentiate into Ts cells, which have a significantly reducedcapacity of producing inflammatory cytokines and have low CD28 and CD40Lexpression, and are thus unable to trigger danger signals from the islettransplant, resulting in the prevention of transplant rejection.

Example 7

Histology: Detection of Transplant Rejection and Graft Versus HostDisease (GVHD)

Islet-engrafted kidneys from humanized NOD/SCID mice (treated withILT3-Fc or IgG) were dissected out and twenty serial paraffin sectionsof were cut at 4-pm thickness. Levels 1, 10, and 20 were stained forlight microscopic evaluation (hematoxylin-eosin). The remaining sectionswere used for immunostains including stains for insulin, CD4 (Biogenics,SanRamon, Calif.), CD3 and CD8 (Dako, Carpinteria, Calif.), and CD40(Abeam, Cambridge, Mass.). Islet quantity and islet lymphocyticinfiltration (insulitis) were graded semi-quantitatively in blindedfashionby a renal pathologist on a scale of 0 to 3+. The degree ofinfiltration by CD8+ T-cells into the islets was graded according to thenumber of CD8+ cells per ×40 high-power field: 0 (none), 1+ (1-10), 2+(11-25), and 3+ (>25). The results were averaged over at least fivehigh-power fields per slide.

Comparison of immuno-stained sections of islet-transplanted kidneys 23days after human PBMC administration (i.e. 30 days post-islettransplantation) showed that islet quantity was greater in theILT3-Fc-treated animal(3+) than human IgG treated control (2+) animal(FIG. 17A-FIG. FIG. 17D). In addition, there was insulitis by CD8+ Tlymphocytes in the control mouse but not in the ILT3-Fc-treated mouse(2+ vs. 0.5+. respectively; FIG. 17E and FIG. 17F).

Pairwise comparison on day 47 showed that islet quantity was greater inILT3-Fc-treated (mean score of 3+) compared to control (mean score of1+) mice (FIG. 19A and FIG. 19B). In addition, slices immunostained withanti-insulin antibodies showed marked reduction of insulin expression byislet P-cells from the IgG treated mouse, indicating transplantrejection (FIG. 20B and FIG. 20D), compared with strong and widespreadexpression in the ILT3-Fc-treated animal (FIG. 20A and FIG. 20C),indicating that the islet transplants were functionally active andwell-tolerated. Insulitis by CD8+ cells was markedly lower inILT3-Fc-treated (mean score 0.5+) vs. IgG-treated control (mean score2.5+) mice (FIG. 19C and FIG. 20D). By light microscopy, islets withinsulitis from control animals exhibited scattered apoptotic bodies.

At 3 months, the tolerated islets in ILT3-Fc-treated mice displayedstrong and diffuse staining for insulin, indicating that the islets werefunctionally active and well tolerated (FIG. 20E). Further, there was alarge quantity of islets (3+) and no insulitis (0; FIG. 20F),demonstrating that the graft was well tolerated.

These histological findings confirm that the IgG-treated host wasundergoing islet allograft rejection, and that administration of ILT3was able to suppress transplant rejection.

Prevention and Treatment of GVHD

Successful engraftment of human PBMC in SCID mice has been known toresult in the development of GVHD, where the implanted T cells mount animmune response against the host, resulting in T-cell expansion andtissue destruction (Hoffmann-Fezer et al., 1993). Animals developingGVHD display symptoms such as hunched back1 lethargy(weight loss andtachypnea. We found that over time1 a portion of hu-NOD/SCID mice(whether they were treated with ILT3-Fc or IgG) developed these symptomsand subsequently died 1 even in mice that maintained viable islet graftsand euglycemic blood glucose levels.

Histological studies confirmed that these mice developed GVHD. Althoughinflammation near the islet graft site can result from transplantrejection1 the mice showing signs of GVHD developed inflammation inareas unconnected to the transplantation site. We observed perivascularinflammation in the kidney contralateral to the one receiving theimplanted islet cells(perivascular and peripelvic inflammation of kidneytissue distant from the implanted islet cells1 and peri-bronchial andperi-vascular inflammation in the lungs.

Comparison of the histological sections between a ILT3-Fc and a IgG(control) treated mouse demonstrated that the GVHD-related symptoms wereless severe in ILT3-Fc treated mouse. Perivascular inflammation wasidentified in the control animal 7 but was not found in the ILT3-Fctreated animal (2+ vs. 0). In the IgG-treated mouse (the Perivascularinflammation involved both the kidney that received islet cell implants(FIG. 17C and FIG. 17D) and the contralateral kidney that did notreceive implants. Examination of hematoxylin and eosin (H&E) stainedsections of the lungs showed diffuse peri-bronchial and peri-vascularinflammation in the IgG treated animal (FIG. 18B and FIG. 18D) whilelung sections from the ILT3-Fc treated animal appeared normal (FIG. 18Aand FIG. 18C). In addition₁ control mice had more pronouncedperivascular and peripelvic inflammation of kidney tissue distant fromthe islets1 with scores ranging from 0-2+ in the ILT3-Fc treated groupto 3+ in the IgG-treated group.

Our results showing that ILT3-Fc treatment suppressed GVHD-specificconditions demonstrate that ILT3-Fc treatment inhibited not only theonset and progression of islet allograft rejection/but also the onset ofGVHD.

In addition, we found that even after excluding the IgG-treated micethat failed to maintain euglycemia, and comparing animals that remainedeuglycemic, host survival time was prolonged significantly in the groupreceiving ILT3-Fc compared to control IgG (FIG. 12). While alleuglycemic mice in the IgG-treated control group (N=8) died within 7weeks1 50% of ILT3-treated mice (8 out of 16) survived for more that 13weeks (p<0.0013) (FIG. 12) demonstrating the effectiveness of ILT3-Fc intreating 20·GVHD.

To further demonstrate the effectiveness of siLT3 in preventing thedevelopment of or treating GVHD, a similar study as above is conducted1without the induction of diabetes or islet cell transplantation. SCIDmice are “humanized” by intraperitoneal (i.p.) injection of 50×106freshly isolated human PBMC and assigned either to the treatment group 1which receives a daily i.p. injection of siLT3 over a period of 10 daysstarting the day of PBMC injection/or to the control group whichreceives human IgG instead.

Host survival time is prolonged significantly in the group receivingILT3-Fc compared to control IgG treatment. While many IgG-treated micedevelop GVHD and die, a significantly larger fraction of ILT3-treatedmice remain free of GVHD and survive through the course of the studyperiod. The average survival time of the ILT3-Fc-treated group issignificantly longer than that of the IgG-treated control group.Subsequent histological studies on the mice show no signs, less severesigns, or a delayed development of GVHD in mice from the ILT3-Fc-treatedgroup compared to controls. Therefore, siLT3 is effective in treating,preventing, or delaying the development of GVHD.

Example 8

ILT3-Fc Inhibits CD40 Signaling in Pancreatic Islet Cells

Pancreatic islet cells express the costimulatory CD40 molecule. Like inAPCs, CD40 expression in cells is upregulated by cytokines and CD40L,and signaling through CD40 activates NF-KB (Klein D, et al., 2005;Cardozo AK et al., 2001). We demonstrated that allospecific,ILT3-Fc-induced CD8+ Ts cells are able to suppress the stimulation ofCD40 expression in islet cells.

Responding T-cells were allostimulated with irradiated PBMC matching theHLA classes I and II of selected islet cultures in the presence of 50pg/ml ILT3-Fc. After 7 days, CD8+ T-cells were isolated and tested.Unprimed CD8+ T-cells from the same responder served as controls.Pancreatic islets selected as targets were co-incubated overnight withone of the following: 1) CD40L-transfected (CD40L+) D1.1 cells only; 2)CD40L+ D1.1 cells plus allospecific CD8+ Ts cells; or 3) CD40L+ D1.1cells plus unprimed CD8+ T-cells. The islet cells, CD8+ T-cells, and theD1.1 cells were added at a 1:1:1 ratio. In addition, islets culturedalone were used to measure the constitutive level of CD40 expression,and islets cultured in tumor necrosis factor-a (10⁶ units/1), IFN-1 (10⁶units/1), and interleukin (IL)-1 (5×104 units/1) were used as a positivecontrol for CD40 induction. After 18 h, cells were washed and theT-cells were depleted. The T-cell depletion was done by magneticseparation, i.e., incubating the cells with mouse anti-CD3 and anti-CD8antibodies (Becton Dickinson, San Jose, Calif.), then incubating with byanti-mouse antibodies fused to magnetic beads (Invitrogen, Carlsbad,Calif.). The cells remaining after magnetic separation, enriched inhuman islet cells, were used for PCR and flow-cytometry studies.

Real time PCR analysis of the isolated islet cells showed (see FIG. 16)that the cytokine mixture induced maximal upregulation of CD40expression in pancreatic islets. CD40L+ D1.1 cells induced thetranscriptional upregulation of CD40, confirming that the presence ofCD40L induces CD40 expression in these islet cells. The presence ofprimed CD8+ Ts cells inhibited the CD40L+ D1.1-induced upregulation ofCD40 to baseline levels (i.e. similar to CD40 expression levels inislets cultured alone). Unprimed CD8+ T cells, however, had no effect oninhibiting CD40 expression in islet cells triggered by CD40L-expressingD1.1 cells. These results demonstrate that allospecific CD8+ Ts cellssuppress CD40L-induced upregulation of CD40 in human pancreatic isletcells.

Prevention or Ttreatment of Autoimmune Destruction of Islet Cells byAdministration of siLT3

Selective autoimmune destruction of native islet cells occursspontaneously or in combination with islet graft rejection in diabeticpatients even while they are receiving immuno-suppressive therapy/andthe discovery of agents that block both of these pathologic processeswould be useful.

The constitutive and selective expression of CD40 on the surface ofcells contribute to autoimmunity and islet allograft rejection byproviding costimulatory signals to the infiltrating lymphocytes. Thecapacity of CD8+ Ts cells to inhibit CD40 signaling in pancreatic cellsis one mechanism underlying Ts-mediated suppression of transplantrejection. In additionl previous work has shown that spontaneousdiabetes in NOD mice is inhibited by treatment with anti-CD40Lantibodies (Balasa Bet al.1 1997) and such treatment also prolongedislet allograft survival in rodent transplantation models (Philips NE etal.1 2003).

We demonstrate that the administration of ILT3-Fc treats diabetes thatarises due to selective autoimmune destruction of native islet cells, byinhibiting the CD40− CD40L interaction between pancreatic islet cells(expressing CD40) and autoaggressive T cells (expressing CD40L) thathave been primed to diabetogenic islet cell peptides presented byself-APC.

To demonstrate the effectiveness of siLT3 in preventing and/or treatingautoimmune diabetes, 3-week-old Non-obese diabetic (NOD) mice aretreated with either ILT3-Fc or IgG. NOD mice are known to spontaneouslydevelop type 1, T cell-dependent autoimmune diabetes, starting fromabout 3-4 weeks postnatal, as a result of insulitis, a leukocyticinfiltration of the pancreatic islets. The NOD mice treated with ILT3-Fcmaintain euglycemia, and show no histological sign of insulitis, whilecontrol mice treated with IgG develop hyperglycemia and insulitis.Treatment of such mice with ILT3-Fc at >9 weeks of age inhibits and/orreverses the disease process. Euglycemia is restored upon treatment withILT3-Fc, and subsequent histology on the mice shows little or no sign ofinsulitis. Therefore, siLT3 is effective in treating diabetes due toselective autoimmune destruction of native islet cells.

CITED DOCUMENTS

-   Leukocyte Immunoglobulin-like Receptor, Subfamily B, Member 4i    LILRB4. OMIM 604821 (2000).-   Beinhauer B. G., et al. (2004) Interleukin 10 regulates cell surface    and soluble LIR-2 (CD85d) expression on dendritic cells resulting in    T cell hyporesponsiveness in vitro. Eur. J. Immunol. 34:74-80.-   Colonna M., et al., U.S. Patent Publication No. 20030165875,    published Sep. 4, 2003.-   Balasa1 B.1 et al. (1997) CD40 ligand-CD40 interactions are    necessary for the initiation of insulitis and diabetes in nonobese    diabetic mice. J. Immunol. 159:4620-4627.-   Cardozo/A. K.₁ et al. (2001) A comprehensive analysis of    cytokine-induced and nuclear factor-kappa B-dependent genes in    primary rat pancreatic beta-cells. J Biol Chern 276:48879-48886.-   Davalli₁ A. M. ₁ et al. (1996) Vulnerability of islets in the    immediate posttransplantation period: Dynamic changes in structure    and function. Diabetes 45:1161-1167-   Gregori₁ 8.₁ et al. (2005) An anti-CD45RO/RB monoclonal antibody    modulates T cell responses via induction of apoptosis and generation    of regulatory T cells. J. Exp. Med. 201:1293-1305.-   Hoffmann-Fezer G.₁ et al. (1993) Immunohistology and immunocytology    of human T-cell chimerism and graft-versus-host disease in SCID    mice. Blood. Jun 15;81(12):3440-3448.-   Jiang/8.8.₁ et al. (1998) Induction of MI-IC-class I restricted    human suppressor T cells by peptide priming in vitro. Hum. Immunol.    59:690-699.-   Klein1 D.₁ et al. (2005) A functional CD40 receptor is expressed in    pancreatic beta cells. Diabetologia 48:268-276.-   Phillips, N. E. , et al. (2003) Blockade of CD40-mediated signaling    is sufficient for inducing islet but not skin transplantation    tolerance. J. Immunol. 170:3015-3023.-   Vlad, G. R., et al. (2005) License to heal: bidirectional    interaction of antigen-specific regulatory T cells and tolerogenic    APC. J. Immunol. 174:5907-5914

What is claimed is:
 1. A method of inhibiting the onset of transplantrejection in a subject who has received, or is about to receive, anislet cell transplant, the method comprising the step of administeringto the subject a therapeutically effective amount of a polypeptidecomprising all or a portion of the extracellular domain of ILT3.
 2. Themethod of claim 1, wherein the polypeptide is administered to thesubject subcutaneously, intradermally, intravenously, orintraperitoneally.
 3. The method of claim 1, wherein the polypeptidefurther comprises a Fc portion of an immunoglobulin, wherein the Fcportion of an immunoglobulin comprises a function-enhancing mutation. 4.The method of claim 3, wherein the Fc portion of an immunoglobulin is aFc portion of IgG
 1. 5. The method of claim 4, wherein the IgG1 is humanIgG1.
 6. The method of claim 3, wherein the function-enhancing mutationin the Fc portion inhibits binding of the Fc portion of animmunoglobulin to an Fc receptor.
 7. The method of claim 6, wherein thefunction-enhancing mutation in the Fc portion is an Asn-->Gln pointmutation at amino acid residue
 77. 8. The method of claim 1, wherein thepolypeptide is administered to the subject concurrently with, or after,islet cell transplantation.
 9. The method of claim 1, wherein thepolypeptide is administered to the subject at onset of islet celltransplant rejection.
 10. The method of claim 1, wherein the subject isa mammal.
 11. The method of claim 10, wherein the subject is a human.12. The method of claim 1, further comprising administering to thesubject an effective amount of one or more additional immunosuppressiveagents.
 13. A method of treating diabetes by inhibiting islet celltransplant rejection in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of apolypeptide comprising all or a portion of the extracellular domain ofILT3.
 14. The method of claim 13, wherein the polypeptide isadministered subcutaneously, intradermally, intravenously, orintraperitoneally.
 15. The method of claim 13, wherein the polypeptidefurther comprises a Fc portion of an immunoglobulin, wherein the Fcportion of an immunoglobulin comprises a function-enhancing mutation.16. The method of claim 15, wherein the Fc portion of an immunoglobulinis a Fc portion of IgG1.
 17. The method of claim 16, wherein the IgG1 isa human IgG1.
 18. The method of claim 15, wherein the function-enhancingmutation in the Fc portion inhibits binding of the Fc portion to an Fcreceptor.
 19. The method of claim 18, wherein the function-enhancingmutation in the Fc portion is an Asn→Gln point mutation at amino acidresidue
 77. 20. The method of claim 13, wherein the polypeptide isadministered to the subject concurrently with, or after, islet celltransplantation.
 21. The method of claim 13, wherein the polypeptide isadministered to the subject at onset of islet cell transplant rejection.22. The method of claim 13, wherein the subject is a mammal.
 23. Themethod of claim 22, wherein the subject is a human.
 24. The method ofclaim 13, further comprising administering to the subject an effectiveamount of another immunosuppressive agent.